The corneal stroma contains collagen fibrils with small uniform diameters and a constant packing, organized into bundles which form orthogonal lamellae. The corneal is unique in being transparent while retaining the mechanical properties to ensure structural integrity. This proposal examines the mechanisms responsible for the assembly of this precisely organized matrix. The hypothesis that collagen type I/V stoichiometry and interactions regulate fibril diameter will be tested. Type V collagen synthesis in 3-D collagen gel cultures will be altered using antisense oligonucleotides followed by biochemical, immunochemical and ultrastructural analyses. Collagen type I/V interactions will be characterized with respect to fibrillar arrangement and the site of initial mixing using electron microscopy with antibodies against defined epitopes. The hypothesis that proteoglycans (Pgs) are most important in the later stages of matrix assembly will be tested. The distribution of PG core proteins will be analyzed using immunoelectron microscopy. The regulation of fibril diameter win be investigated using antisense oligonucleotides and other inhibitors of PG synthesis. The regulation of matrix assembly will be evaluated in ovo after infection with retroviral vectors expressing antisense RNA to inhibit PG synthesis. The hypothesis that type VI collagen has a role in cell-matrix and matrix-matrix interactions will be addressed by defining corneal fibroblast-type VI collagen interactions using in vitro assays. The matrix receptors responsible for these interactions will be identified and interactions of type VI collagen with other macromolecules will be evaluated using binding assays. The hypothesis that cell-matrix interactions are important in defining the migratory pathway into the cornea and providing cues for corneal differentiation will be tested. The matrix molecules in areas through which the presumptive corneal fibroblasts migrate will be identified immunocytochemically. Cell interactions and migration on these components will be examined in vitro. The influence of the primary stroma, epithelium and matrix components on differentiation will be assessed. The collagen type I/V phenotype and antibodies against corneal stroma-specific epitopes will be used as markers for differentiation. These studies will contribute to the elucidation of the mechanisms involved in the assembly of tissue-specific extracellular matrices. Knowledge of matrix-matrix and cell-matrix interactions is essential for an understanding of development, growth, repair and transparency.